Bacteria often export high molecular weight polysaccharides that can form a capsule associated with the bacterial surface, or be secreted into the surrounding environment as exopolysaccharide. Capsules can help bacteria evade host immune systems, promote adhesion to host cells, and form biofilms. Like lipopolysaccharide (LPS), group 1 and 4 capsules are assembled from short oligosaccharide repeat units by a Wzy-like glycosyltransferase, but are typically much larger (>500 kDa). Gram-negative bacteria require additional molecular machinery to transport the capsule polysaccharide through the outer membrane where it associates with the LPS by an unknown mechanism. The current model is that Wza, a conserved outer membrane auxiliary protein necessary for capsule production, forms an octamer with a helical pore in the outer membrane from which the polysaccharide chain exits. But are there other proteins that could function in this role? Enteropathogenic and enterohaemorrhagic Escherichia coli require the seven genes of the gfc operon to produce a group 4 capsule comprised of O-antigen repeating units. Unlike the group 1 system, there are four essential genes (gfcABCD) upstream of gfcE (Wza homolog) that encode periplasmic or outer membrane proteins of unknown function. Interestingly, E. coli and many other Gram-negative species encode a similar yjbEFGH operon implicated in biofilm formation and group 1 capsule expression. GfcD and YjbH are predicted to be large outer membrane 2-barrel proteins that also may be acylated. The overall hypothesis is that this protein is part of a more complex secretion system that provides an exit route for the polysaccharide itself or for an accessory molecule through the outer membrane. To clarify the structure and role of GfcD in polysaccharide export we propose two aims for this R21. In Specific Aim 1, experiments will confirm the membrane location of GfcD, characterize the degree to which a gfcD- mutant affects capsule expression in vivo, and identify what other proteins interact with GfcD. In Specific Aim 2, we will prepare expression constructs of GfcD and three homologs and evaluate which will be best suited to for large scale purification. Proteins will be screened with a variety of detergents for optimal solubility and monodispersity. Pure protein will be used to screen for crystallization conditions. Labeled protein will also be prepared for solid-state NMR experiments to examine protein orientation, secondary structure and for the presence of a solvent accessible channel. Results from this short-term project will provide the foundation for a longer term project to determine the GfcD structure, elucidate the function of the other Gfc proteins in polysaccharide export, and evaluate if the export system could be a target for broad spectrum drugs aimed to suppress capsule expression. PUBLIC HEALTH RELEVANCE: Enteropathogenic E. coli (EPEC) and other bacteria are surrounded by a capsular polysaccharide that is often important for pathogenicity or biofilm formation. EPEC GfcD is a putative outer membrane 2-barrel protein conserved in at least 50 bacterial genera and implicated in capsule expression. We propose to characterize the role of the protein in polysaccharide export, and develop expression systems for NMR and crystallographic analyses.